Quantitative Microscopy: FLIM, FRET, FCS 

FLIM (Fluorescence Lifetime Imaging Microscopy) provides additional information beyond fluorescence intensities by measuring fluorescence lifetimes. This enables applications such as monitoring cellular metabolism, characterizing the local molecular environment (e.g. temperature, viscosity, pH, and ion concentration), and distinguishing fluorescent species with overlapping emission spectra. More information can be found in the following review.

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FRET (Förster Resonance Energy Transfer) enables the detection and quantification of molecular interactions occurring within distances of up to approximately 10 nm, forming the basis of numerous biosensor applications. Combining FLIM with FRET (FLIM-FRET) improves the accuracy and robustness of these measurements by making them independent on fluorescence intensity variations. Both FLIM and FRET are compatible with widefield, confocal, multiphoton and light-sheet microscopies.

FCS (Fluorescence Correlation Spectroscopy) analyzes fluorescence intensity fluctuations generated by the diffusion of fluorescent molecules through the focal volume. From these fluctuations, parameters such as diffusion coefficients, molecular concentrations, and co-diffusion can be quantitatively determined. More information can be found in the following review